A Study On Some Basic Problems Of Polycation Gene Carriers By Chitosan, PEI And Their Derivatives

Polycations as gene carriers have attracted an increasing number of attentions in the past decade due to the increase of inherent diseases and malignant diseases. With positive charge, polycations can steadily form complexes (so-called’polyplex’) with negatively charged DNA, and then these polyplexes can be used for gene transfection. Compared with viral vectors, the advantages of polycations include construction flexibility, facile fabrication and low immunogenicity, etc., although the transfection efficiency (TE) mediated by polycations is relatively low. The polycations used for gene carriers in literature commonly involve two categories, i.e. synthetic polymers (such as PEI) and natural derived macromolecules (such as chitosan). However, a few of fundamental issues such as the mechanism of polycation mediated gene transfection etc. are still unclear. In the present study, we tried to deeply understand some of these open questions, with details below:1. Investigation of the factors affecting the relationship between chain length and TE (RCL-TE) using 0-methyl free N,N,N-trimethyl chitosan (TMC) as gene carriers. Longer chain length polycations are generally believed to mediate higher TE. However, for chitosan and its derivatives (such as TMC), no consensus has been obtained on the RCL-TE.Hereby, we synthesized four 0-methyl free TMCs with various chain lengths via regioselective N,N,N-trimethylation avoiding O-methylation. These TMCs have well-defined structure and are used as gene carriers. It was found that longer chain length TMC mediated higher luciferase expression in COS-7 cell, 293T cell and NCI-H1299 cell, and mediated higher enhanced green fluorescence protein (EGFP) expression in 293T cell, indicating longer chain length are benefit for efficient gene transfection. Thus, TMC is not an exception of longer chain length polycations mediated higher TE. Then various factors were investigated to ascertain the key ones in RcL-TE.The results showed that the key factors include polyplex stability, cellular uptake pathway, and intracellular trafficking, rather than polycation-DNA binding affinity, polyplex particle size in water, zeta potential, serum, cytotoxicity, and cellular uptake efficiency. It should be noted that some factors, such as cytotoxicity and cellular uptake efficiency, are commonly believed to influence the transfection performance. But here, these factors are evidently not the key ones in RcL-TE.For rational design of polycations, especially chitosan-based polycations as gene carriers, polycations with relative long chain lengths are more favorable and more attention should be paid to polyplex stability, function of uncomplexed polycation chains, cellular uptake pathway, and intracellular trafficking.2. Evaluation of the optimal N/P ratios (mole ratio of the nitrogen in polycation to the phosphorous in DNA) in serum-free or serum-containing DMEM using branched PEI and its derivatives as gene carriers, and investigation of other factors possibly affect TE. In the literatures, the TE mediated by polycations is commonly evaluated at some designated N/P ratios under various conditions. Actually, the optimal N/P ratios may vary with the test conditions changing. Thus, it is not reasonable to use the same N/P ratios under different conditions. In this study, PEI and its derivatives (methylated PEI, quaternized PEI, acetylated PEI, Alanine modified PEI, Lysine modified PEI, guanidinated PEI, PEGylated PEI, and imidazole modified PEI and so on) were used to investigate this issue. The results showed that the optimal N/P ratios of PEI derivatives are different from their original PEI. For example, the optimal N/P ratios of the original PEI were 6-10, while those of Alanine modified PEI were 40-80, and PEGylated PEI were no lower than 100. In the presence of serum (10 v%), the optimal N/P ratios were much higher than those in the absence of serum. For instance, the optimal N/P ratios of the original PEI, Alanine modified PEI and PEGylated PEI were 20-30, no lower than 100, and no lower than 250, respectively. Besides serum, other factors, such as conditions to prepare polyplex, dose of polyplex, additives in cell culture media, and cell lines employed to test TE, may affect the transfection and optimal N/P ratios. Thus, these factors should be noted when evaluating polycations as gene carriers, especially when using PEI as control, the optimal N/P ratios should be verified and adopted.3. Investigation of the function of polycations free chain using TMC/pDNA polyplex (TMC-P) plus PEI free chain (PEI-F) as gene carriers. Generally, polyplexes can be formed at low N/P ratios, but efficient gene transfection is only archived at higher N/P ratios. Therefore, the polycation free chains play a crucial role in efficiency gene transfection. When evaluating the function of polycations free chain, PEI-P plus PEI-F are not a good system because PEI-P is not stable in DMEM and it is hardly to exclude the interaction between PEI-P and PEI-F. On the other hand, TMC-P plus PEI-F are quite stable in DMEM, and the polycations in polyplex and free in solution are different, TMC-P plus PEI-F are much more suitable to investigate the function of polycation free chain. By testing the luciferase expression of TMC-P plus PEI-F, PEI-F was found to evidently improve the TE of TMC-P, indicating the polycations free chain functions significantly in the process of gene transfection. By testing the gene expression in the presence of inhibitors of cellular uptake pathways, the polycation free chain was found to affect the cellular uptake pathways of polyplexes. Meanwhile, the polycations free chain may affect the intracellular trafficking of polyplexes. Nevertheless, the details of the function of polycation free chain still require further investigation, and the polycation free chain could be used for polymer therapy.Obviously, gene expression mediated by polycations is an extremely complicate process. Dedicated design is required in the whole process, from the synthesis of polycations, to the preparation of polyplexes, next to the delivery of polyplexes, then to the recognition and internalization by the targeted cells, then to the intracellular trafficking, finally to the nucleus and function there. Fundamentally understanding of polycations as gene carriers is essential to prepare an efficient gene carrier. The results in the present study provides insight into rational design and evaluation of polycations as gene earriers.